This invention relates to the treatment of bleeding disorders. In particular, this invention relates to the use of tissue factor protein to effect haemostasis in certain clinical conditions and particularly in animals lacking certain coagulation proteins. Factor VIII and factor IX deficiencies are two examples.
Bleeding is one of the most serious and significant manifestations of disease. It may occur from a local site or may be generalized. Bleeding associated with a local lesion may be superimposed on either a normal or a defective haemostatic mechanism. Normal haemostasis comprises mechanisms operative immediately following an injury and those acting over a longer period. Primary haemostasis consists principally of two components: vasoconstriction and platelet plug formation. The maintenance mechanism consists of the fibrin clot produced by the coagulation system. Platelet plug formation is especially important in capillary haemostasis, while vasoconstriction and fibrin clot formation is more important in larger vessel haemostasis. In the microcirculation haemostasis consists of asoconstriction and platelet plug formation. Platelet plug formation may be divided into several stages: adhesion of platelets to subendothelial surfaces exposed by trauma; platelet activation release reaction; platelet aggregation, which results in the sequestration of additional platelets at the site, and the binding of fibrinogen and the coagulation proteins to the platelet surface which includes thrombin formation; and, fusion which is the coalescence of fibrin and fused platelets to form a stable haemostatic plug.
Blood coagulation performs two functions; the production of thrombin which induces platelet aggregation and the formation of fibrin which renders the platelet plug stable. A number of discrete proenzymes and procofactors, referred to as "coagulation factors", participate in the coagulation process. The process consists of several stages and ends with fibrin formation. Fibrinogen is converted to fibrin by the action of thrombin. Thrombin is formed by the proteolytic cleavage of a proenzyme. prothrombin. This proteolysis is effected by activated factor X (referred to as factor X.sub.a) which binds to the surface of activated platelets and in the presence of Va and ionic calcium cleaves prothrombin.
Activation of factor X may occur by either of two separate pathways, the extrinsic or the intrinsic (FIG. 1). The intrinsic cascade consists of a series of reactions wherein a protein precursor is cleaved to form an active protease. At each step, the newly formed protease will catalyze the activation of the precursor protease at the subsequent step of the cascade. A deficiency of any of the proteins in the pathway blocks the activation process at that step, thereby preventing clot formation and typically gives rise to a tendency to hemorrhage. Deficiencies of factor VIII or factor IX, for example, cause the severe bleeding syndromes haemophilia A and B, respectively In the extrinsic pathway of blood coagulation, tissue factor, also referred to as tissue thromboplastin, is released from damaged cells and activates factor X in the presence of factor VII and calcium. Although activation of factor X was originally believed to be the only reaction catalyzed by tissue factor and factor VII, it is now known that an amplification loop exists between factor X, factor VII, and factor IX (Osterud, B., and S. I. Rapaport, Proc. Natl. Acad. Sci. USA 74:5260-5264, 1977; Zur, M. et al., Blood 52: 198, 1978). Each of the serine proteases in this scheme is capable of converting by proteolysis the other two into the activated form, thereby amplifying the signal at this stage in the coagulation process (FIG. 2). It is now believed that the extrinsic pathway may in fact be the major physiological pathway of normal blood coagulation (Haemostasis 13: 150-155 1983). Since tissue factor is not normally found in the blood, the system does not continuously clot; the trigger for coagulation would therefore be the release of tissue factor from damaged tissue.
Tissue factor is an integral membrane glycoprotein which. as discussed above, can trigger blood coagulation via the extrinsic pathway. Bach, R. et al., J. Biol. Chem. 256(16), 8324-8331 (1981). Tissue factor consists of a protein component (previously referred to as tissue factor apoprotein-III) and a phospholipid. Osterud, B. and Rapaport, S. I., PNAS 74, 5260-5264 (1977). The complex has been found on the membranes of monocytes and different cells of the blood vessel wall. Osterud, B., Scand J. Haematol. 32, 337-345 (1984). Tissue factor from various organs and species has been reported to have a relative molecular mass of 42,000 to 53.000. Human tissue thromboplastin has been described as consisting of a tissue factor protein inserted into phospholipid bilayer in an optimal ratio of tissue factor protein:phospholipid of approximately 1:80 Lyberg, T. and Prydz. H., Nouv. Rev Fr. Hematol 25(5), 291-293 (1983). Purification of tissue factor has been reported from various tissues such as: human brain (Guha, A. et al. PNAS 83, 299-302 [1986]and Broze, G. H. et al., J. Biol. Chem. 260[20], 10917-10920 [1985]); bovine brain (Bach, R. et al., J. Biol. Chem. 256, 8324-8331 [1981]); human placenta (Bom, V. J. J. et al., Thrombosis Res. 42:635-643 [1986]; and. Andoh, K. et al., Thrombosis Res. 43:275-286 [1986]); ovine brain (Carlsen, E. et al., Thromb. Haemostas. 48[3], 315-319 [1982]); and, lung (Glas, P. and Astrup, T., Am J. Physiol. 219, 1140-1146 [1970]. It has been shown that bovine and human tissue thromboplastin are identical in size and function. See for example Broze, G. H. et al., J. Biol. Chem. 260(20), 10917-10920 (1985) It is widely accepted that while there are differences in structure of tissue factor protein between species there are no functional differences as measured by in vitro coagulation assays. Guha et al. supra. Furthermore, tissue factor isolated from various tissues of an animal, e.g. dog brain, lung, arteries and vein was similar in certain respects such as, extinction coefficient, content of nitrogen and phosphorus and optimum phospholipid to lipid ratio but differed slightly in molecular size, amino acid content, reactivity with antibody and plasma half life. Gonmori. H. and Takeda, Y., J. Physiol. 229(3), 618-626 (1975). All of the tissue factors from the various dog organs showed clotting activity in the presence of lipid. Id. It is widely accepted that in order to demonstrate biological activity, tissue factor must be associated with phospholipids. Pitlick. F. A., and Nemerson. Y., Biochemistry 9, 5105-5111 (1970) and Bach, R. et al. supra. at 8324. It has been shown that the removal of the phospholipid component of tissue factor, for example by use of a phospholipase, results in a loss of its biological activity. Nemerson, Y., J.C.I. 47, 72-80 (1968). Relipidation can restore in vitro tissue factor activity. Pitlick, F. A and Nemerson, Y. Biochemistry 9, 5105-5113 (1970) and Freyssinet, J. M. et al., Thrombosis and Haemostasis 55, 112-118 [1986].
Infusion of tissue factor has long been believed to compromise normal haemostasis. In 1834 the French physiologist de Blainville first established that tissue factor contributed directly to blood coagulation. de Blainville H. Gazette Medicale Paris, Series 2, 524 (1834). de Blainville also observed that intravenous infusion of a brain tissue suspension caused immediate death which he observed was correlated with a hypercoagulative state giving rise to extensively disseminated blood clots found on autopsy. It is now well accepted that intravenous infusion of tissue thromboplastin induces intravascular coagulation and may cause death in various animals. (Dogs: Lewis, J. and Szeto I. F., J. Lab. Clin. Med. 60, 261-273 (1962); rabbits: Fedder. G. et al., Thromb. Diath. Haemorrh. 27, 365-376 (1972); rats: Giercksky, K. E. et al., Scand. J. Haematol. 17 305-311 (1976); and, sheep: Carlsen, E. et al., Thromb Haemostas. 48, 315-319 [1982]).
In addition to intravascular coagulation or a hypercoagulative state resulting from the exogenous administration of tissue factor, it has been suggested that the intravascular release of tissue thromboplastin may initiate disseminated intravascular coagulation (DIC). Prentice, C. R., Clin. Haematol. 14(2), 413-442 (1985). DIC may arise in various conditions such as shock, septicaemia, cardiac arrest, extensive trauma, bites of poisonous snakes, acute liver disease, major surgery, burns, septic abortion, heat stroke, disseminated malignancy, pancreatic and ovarian carcinoma, promyelocytic leukemia, myocardial infarction, neoplasms, systemic lupus erythematosus, renal disease and eclampsia. Present treatment of DIC includes transfusion of blood and fresh frozen plasma; infusion of heparin; and removal of formed thrombi. The foregoing clinical syndromes suggest that endogenous release of tissue factor can result in severe clinical complications. Andoh, K. et al., Thromb. Res 43, 275-286 (1986). Efforts were made to overcome the thrombotic effect of tissue thromboplastin using the enzyme thromboplastinase. Gollub, S. et al., Thromb. Diath. Haemorh. 7, 470-479 (1962). Thromboplastinase is a phospholipase and would presumably cleave the phospholipid portion of tissue factor. Id.
Congenital disorders of coagulation characteristically involve a single coagulation protein. Haemophilia is a bleeding disorder due to inherited deficiency of a coagulation factor. e.g. the procoagulant activity of factor VIII. The basis of therpay of bleeding episodes is transfusion of material containing the missing coagulation protein, e.g. infusion of factor VIII procoagulant activity which temporarily corrects the speicific defect of haemophilia A.
Von Willebrand's disease is another bleeding disorder characterized by a prolonged bleeding time in association with an abnormality or deficiency in the von Willebrand protein. Treatment is by infusion of normal plasma or by a composition rich in von Willebrand protein. Congenital deficiencies of each of the other coagulation factors occur and may be associated with a haemorrhagic tendency. The present therapies for the deficiencies are: factor IX deficiency is treated using concentrates containing factor IX: infusions of plasma are given for a factor XI deficiency; and plasma infusion is given for a factor XIII deficiency.
Acquired coagulation disorders arise in individuals without previous history of bleeding as a result of a disease process. Inhibitors to blood coagulation factors may occur in multitransfused individuals. Acquired coagulation factor deficiencies with unknown etiology also give rise to haemostatic problems. DIC describes a profound breakdown of the haemostatis mechanism.
An object of the present invention is to provide a coagulation inducing therapeutic composition for various chronic bleeding disorders, characterized by a tendency toward hemorrhage, both inherited and acquired Examples of such chronic bleeding disorders are deficiencies of factors VIII. IX, or XI. Examples of acquired disorders include: acquired inhibitors to blood coagulation factors e g, factor VIII, von Willebrand factor, factors IX, V, XI, XII and XIII; haemostatic disorder as a consequence of liver disease which includes decreased synthesis of coagulation factors and DIC; bleeding tendency associated with acute and chronic renal disease which includes coagulation factor deficiencies and DIC; haemostasis after trauma or surgery; patients with disseminated malignancy which manifests in DIC with increases in factors VIII, von Willebrand factor and fibrinogen; and haemostasis during cardiopulmonary surgery and massive blood transfusion. Another object of this invention is to provide a method of treatment of such chronic bleeding disorders.
A further object of this invention is to provide a coagulation inducing therapeutic composition for acute bleeding problems in normal patients and in those with chronic bleeding disorders. Another object of this invention is to provide a method of treatment for such acute bleeding problems.
Yet another object of this invention is to provide an anticoagulant therapeutic, that is an antagonist to tissue factor protein, to neutralize the thrombotic effects of endogenous release of tissue thromboplastin which may result in a hypercoagulative state. Particularly, such an anticoagulant, that is an antagonist to tissue factor protein, would neutralize the hypercoagulant effects of endogenously released tissue thromboplastin by inactivating tissue factor protein. Such a tissue factor protein antagonist can be an antibody or other protein that specifically inactivates the protein component.